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Removing Exonuclease From Protein Prep - How can I remove exonuclease from a protein prep (Mar/08/2005 )

Can anybody help me with this? I'm trying to purify a protein that has DNA as substrate, but I cannot seem to purify away the exonuclease. So when I try to assay for my protein, the DNA substrate is just chew up very fast. However, the SDS gel showed the protein prep was very clean. I use a NiNTA and size exclusion for purification. Also tried anion or cation exchange, which doesn't get rid of the exo's completely. What can I do to get rid of these exo activity completely? Is there any exonuclease-minus strain that I can use for over expression of my protein?

Thanks

-chemgirl-

QUOTE (chemgirl @ Mar 8 2005, 03:11 PM)
Can anybody help me with this? I'm trying to purify a protein that has DNA as substrate, but I cannot seem to purify away the exonuclease.  So when I try to assay for my protein, the DNA substrate is just chew up very fast.  However, the SDS gel showed the protein prep was very clean.  I use a NiNTA and size exclusion for purification.  Also tried anion or cation exchange, which doesn't get rid of the exo's completely.  What can I do to get rid of these exo activity completely?  Is there any exonuclease-minus strain that I can use for over expression of my protein?

Thanks



I have the same problem. Can anyone help?


wes

-wes-

is your protein expressed as a fusion protein?

also, silver staining can show things on your gel you would never see with coomasie.

-chempilot-

QUOTE (wes @ Aug 30 2005, 04:44 PM)
QUOTE (chemgirl @ Mar 8 2005, 03:11 PM)
Can anybody help me with this? I'm trying to purify a protein that has DNA as substrate, but I cannot seem to purify away the exonuclease.  So when I try to assay for my protein, the DNA substrate is just chew up very fast.  However, the SDS gel showed the protein prep was very clean.  I use a NiNTA and size exclusion for purification.  Also tried anion or cation exchange, which doesn't get rid of the exo's completely.  What can I do to get rid of these exo activity completely?  Is there any exonuclease-minus strain that I can use for over expression of my protein?

Thanks



I have the same problem. Can anyone help?


wes

Hi chempilot:

My protein is a 6xHis-tagged thioredoxin fusion protein. I think most people do not use their purified proteins in experiemnts involving DNA or RNA and that could be why very few people have noticed that their so-called purified proteins actually contain some nuclease activity (they don't have to care about it anyway). Novagen says that Benzonase (a nuclease used to prepare cell lysate beofre purification) should not be used if your proteins need to be nuclease-free. I did not use any nuclease to prep my lysate but I still can see nuclease activity (my protein shouldn't have any nuclease activity) in my very pure protein prep. My question is, is there a realtively easy and efficient way to get ride of the trace nuclease contamination from a seemingly very pure protein prep? Any one, please?

wes



-wes-

i also use 6His thioredoxin tagged proteins and was trying to remove all ATPase contamination for an assay i would perform on the native protein. i would perform the anion exchange and size exclusion after cutting and found i would always have contamination.

i figured out that if i performed my anion exchange before cutting the tag off, i could use the tag as an added property of the protein (i.e. different size, shape, charge etc.) than my native protein without the tag to separate away the ATPases.

after doing this my ATPase problem vanished! hope this helps!

-chempilot-